Toxins and antibodies of C. difficile

ABSTRACT

Mono-specific antibodies for each of toxin A and toxin B of C. difficile are produced and used in an assay for toxin A and toxin B, respectively. Purified toxin A of C. difficile is also produced.

This invention relates to a C. difficile and more particularly to theproduction of antibodies to the toxins of C. difficile, purification ofthe toxins, and the use thereof in an assay for C. difficile toxins.

The anaerobic organism Clostridium difficile (C. difficile) isassociated with antibiotic related pseudomembranes colitis and as aresult, there have been tests developed to ascertain the presence of C.difficile antigen in specimens of human stool.

One such test involves culture of human feces, which requiresspecialized facilities and a long period of time. This test also detectsstrains of C. difficile that do not produce toxins, and thus gives falsepositive results.

Another test involves counter immunoelectrophosesis, however, this test,as currently used, is not sensitive enough to detect toxins and gives alot of false positives.

A further test involves an enzyme immunoassay; however, such test, ascurrently used, does not differentiate between toxic and non-toxicstrains, and as a result, the test may give misleading results.

The present invention is directed to antibodies for toxins of C.difficile, toxins of C. difficile, and an assay for toxigenic C.difficule.

In accordance with one aspect of the present invention, there isprovided a mono-specific antibody for toxin A of C. difficile, and suchmono-specific antibody supported on a solid support.

In accordance with another aspect of the present invention, there isprovided a mono-specific antibody for toxin B of C. difficile, and suchmono-specific antibody supported on a solid support.

In accordance with a further aspect of the present invention, there isprovided pure toxin A of C. difficile, and such toxin on a solidsupport.

In accordance with yet another aspect of the present invention, there isprovided an assay for toxin A of C. difficile which uses mono-specificantibody for toxin A.

In accordance with yet a further aspect of the invention, there isprovided an assay for toxigenic C. difficile.

The term "mono-specific antibody for toxin A", as used herein, means anantibody which does not have any determinant sites for antigens of C.difficile other than toxin A.

The term "mono-specific antibody for toxin B", as used herein, means anantibody which does not have any determinant sites for antigens of C.difficile other than toxin B.

The term mono-specific antibody as used herein includes such antibody ina mono-clonal form.

It is to be understood that the mono-specific antibodies to toxin Aand/or toxin B can be produced from an organism other than C. difficile,so long as the antibody does not have a determinant site for anotherantigen of C. difficile.

C. difficile antibody or antibody to C. difficile means antibody whichis not mono-specific, and which therefore is comprised of a mixture ofantibodies, which includes antibodies for toxins of C. difficile(antibody for toxin A and antibody for toxin B) and antibodies fornon-toxins of C. difficile.

Antibody for toxigenic C. difficile or antibody specific for toxins ofC. difficile means antibody which does not have determinant sites forantigens of C. difficile other than toxin A and toxin B (a mixture ofantibody specific only for toxin A and antibody specific only for toxinB).

Toxin A is the C. difficile toxin that is generally referred to as theenterotoxin. Toxin A has a native molecular weight between 550,000 and600,000, an isoelectric point of 5.5, contains no detectablecarbohydrate or phosphorus and does not exhibit detectable proteaseacctivity. It is inactivated as pH 2.0 and is stable at a pH of 10.0.Toxin A is eluted from DEAE by a buffer containing 0.16M NaCl.

Toxin B is the C. difficile toxin which is generally referred to as thecytotoxin of C. difficile. Toxin B has a native molecular weight between380,000 and 470,000, an isoelectric point of 3.8; contains no detectablephosphorus, but does contain very small amounts of carbohydrate (whichmay be a contaminant) and does not exhibit detectable protease activity.Toxin B is inactivated by pH's less than 2.0 and over 10.0, and iseluted from a DEAE column with a salt concentration of 0.4M.

It is to be understood, however, that although toxin A is referred to asthe enterotoxin such toxin A also has cytotoxicity.

In accordance with one aspect of the invention, toxin A, which has beenpartially purified by separation from toxin B, and which still includessome nontoxigenic proteins is further purified to produce pure toxin A.In accordance with this aspect of the invention, the pH and molarity ofan aqueous solution of toxin A are adjusted to precipitate toxin A,without precipitating the remaining proteins, whereby pure toxin A isrecovered.

More particularly, the pH of the aqueous solution is adjusted to a pH ofless than 6.0 and at which toxin A precipitates without precipitation ofother proteins or denaturation of the toxin, and the molarity of theaqueous solution is adjusted to less than 0.1M and at which toxin Aprecipitates without precipitation of other proteins. In general the pHis at least 5.0, with the pH preferably being from 5.3 to 5.7, with thebest results being achieved at pH 5.5. The molarity of the solution isgenerally at least 0.001M, with best results being achieved at 0.01M.

The molarity and pH may be achieved by using a suitable salt buffer;e.g., a sodium acetate buffer. The adjustment of molarity may beconveniently achieved by dialysis, although other procedures areapplicable.

The precipitated pure toxin A is recovered from the aqueous solution andmay be solubilized in water at a buffered pH of about 7.5.

The partially purified toxin A, which is purified in accordance with theinvention to produce pure toxin A may be recovered by proceduresgenerally known in the art. For example, the supernatent from a cellculture of a toxigenic C. difficile strain is concentrated with anultrafiltration membrane that retains only large molecules (over 100,000M.W.) and the retained material is applied to a chromatographic column.The column (DEAE) is then eluted with gradients of sodium chloride (thefirst gradient is 0.05-0.25M NaCl with a 0.3M NaCl wash and the secondgradient is 0.3-0.6 NaCl), with the first gradient eluting toxin A andthe second gradient toxin B.

The term "pure toxin A" as used herein, indicates that the toxin Apreparation is free of contaminating substances (only toxin A ispresent) when examined by a variety of highly resolving techniques knownin the art. The term partially purified, as used herein, indicates thatsome contaminants, but not all, have been removed. Pure toxin A whenprepared by the procedures described above is pure by the criteria of: asingle band on acrylamide gel electrophoresis when done with 100 ug ofprotein per gel rod (Davis, SDS, and gradient gels); a singleimmunoprecipitin arc on crossed immunoelectrophoresis plates withantisera made to the complete mixture of C. difficile antigens; and puretoxin A when injected into animals elicits production of a mono-specificantibody to toxin A.

The mono-specific antibody for toxin A of C. difficile and themono-specific antibody for toxin B of C. difficile may be prepared byseveral different procedures.

In accordance with one procedure, C. difficile culture supernatantfluids produced by a known cultivating procedure are boiled to destroyall heat-labile protein antigens (toxin and non-toxin antigen) andthereby provide material containing only the heat-stable antigens of C.difficile. These antigens are then supported on a first cyanogen bromideactivated Sepharose column.

Partially purified toxin A and partially purified toxin B, each obtainedby elution from a DEAE chromatographic column, as hereinabove described,are coupled to a second and third cyanogen bromide activated Sepharosecolumn, respectively.

Antibodies to crude C. difficile antigens toxin (such toxin includesboth toxin A and toxin B as well as many other antigens produced by thebacterium) are produced in a suitable animal; e.g. goats, and theelicited antibody is comprised of an antibody mixture to C. difficileantigens (such antibody mixture includes antibodies for toxin A andtoxin B, as well as antibodies to the non-toxin antigens, includingantibodies to the heat-stable antigens.) The non-toxin antibodies(except the antibodies to the non-toxin heat-stable antigens) areremoved from the antibody mixture by contact with whole cells of anon-toxic strain of C. difficile to thereby bind the antibodies tonon-toxins except for the antibodies to the nontoxic heat-stableantigens.

Subsequently, the antibody mixture (which now contains the antibodiesfor the toxins, and the antibodies to the non-toxic heat-stableantigens) is then applied to the first column on which the heat-stableantigens of C. difficile are supported, whereby the sugar antibodies tothe heat-stable antigens become bound.

The mixture which is free of antibody against the heat-stable antigensand contains antibodies to toxins A and B is then divided into twoparts, with one part being applied to the second column on which puretoxin A is supported, and the other part being applied to the thirdcolumn on which partially purified toxin B is supported, whereby in thesecond column, the antibody to toxin A becomes selectively bound to thesupported toxin A and in the third column, the antibody to toxin Bbecomes selectively bound to the supported toxin B.

The antibodies for toxin A and the antibodies for toxin B are eachsubsequently eluted from the second and third columns, respectively;e.g., by the use of potassium thiocyanate to thereby, respectively,produce mono-specific antibody for toxin A and mono-specific antibodyfor toxin B.

In some cases as hereinafter described, the mixture of antibody fortoxin A and antibody for toxin B (after removal of non-toxic antigens)may be used without separation into mono-specific antibody for each ofthe toxins, e.g., in an assay for toxigenic C. difficile.

Alternatively, mono-specific antibody to toxin A may be produced byapplying the crude C. difficile antibody onto a column support withimmobilized pure toxin A. The non-toxin A antibodies are removed fromthe column by extensive washing and the remaining antibodies, which areattached to the toxin A, are eluted with potassium thiocyanate.

Alternatively, mono-specific antibody to toxin A may be produced frompurified toxin A, prepared as hereinabove described, by injecting toxinA (mixed with some formaldehyde to increase toxicity without destroyingantigenicity or neutralized with antibody) into a suitable animal; e.g.a goat. The mono-specific antibody to toxin A is then recovered by theprocedure described in the preceding paragraph.

The mono-specific antibodies and the toxins of the present invention maybe supported on a solid support for use in an assay for C. difficile.Alternatively, such antibodies and toxins may be used in such an assayin an unsupported form.

In using a solid support, the solid support may be any of a wide varietyof solids, and may be employed in any one of a wide variety of forms;e.g. plates, trays, particles, tubes, sheets, etc.

As representative examples of suitable supports, there may be mentioned:synthetic polymer supports, such as polystyrene, polypropylene,substituted polystyrene (e.g. animated or carboxylated polystyrene),polyacrylamides, polyamides, polyvinylchloride, etc.; glass beads,agarose; etc. The supports may include reactive groups, e.g. carboxylgroups, amino groups, etc. to permit direct linking to the support.

The antibodies and toxins of the present invention may be supported on asolid support in a variety of ways; for example, by adsorption, covalentcoupling, activation of a suitable support, with protein A, etc.

As representative examples of suitable coupling agents there may bementioned: dialdehydes; for example glutaraldehyde, succinaldehyde,malonaldehyde, etc.; unsaturated aldehyde, e.g., acrolein, methacrolein,crotonaldehyde, etc.; carbodiimides, diisocyanates; dimethyladipimate;cyanuric chloride etc. The selection of a suitable coupling agent shouldbe apparent to those skilled in the art from the teachings herein.

Similarly, the antigen may be supported by activation of a suitablesupport; for example, cyanogen bromide activated agarose.

In accordance with an aspect of the present invention, the antibodiesand toxins of the present invention may be used in an assay for eithertoxin A, or toxin B of C. difficile or for toxigenic C. difficile (bothtoxin A and toxin B).

In some of such assays, one or more of such substances are used in a"labelled" or "tagged" form, and such labels or tags are of a type knownin the art for use in assays. Thus, for example, the label or tag may bea radioactive substance, such as radioactive iodine, radioactive cobalt,tritium, etc.; an enzyme; a fluoroescent material; a chemiluminescentmaterial, etc.

The labels may be added to the various substances by procedures asgenerally practiced in the art. Similarly, the label or tag may bedetected by procedures known in the art; for example, counters forradioactive labels, colorimetric detection of enzymes, etc.

The antibodies and toxins of the present invention may be used insupported and/or unsupported form for the assay of C. difficile.

In accordance with one embodiment of the invention, there is provided anassay for toxin A of C. difficile by use of the mono-specific antibodyfor toxin A.

In accordance with one aspect of this embodiment, antibody to C.difficile is supported on a solid support; for example, a microtiterplate. The supported C. difficile antibody is then contacted with asample to be analyzed (analyte) such as a dilution of patient feces, andas a result of such contact, any toxin A present in the analyte, as wellas other antigens of C. difficile, become bound to the supported C.difficile antibody. Subsequently, the bound analyte portion is contactedwith mono-specific antibody for toxin A of C. difficile, (raised in ananimal different than the animal in which C. difficle antibody wasraised,) and such mono-specific antibody is only bound by any toxin Apresent in the bound analyte portion.

This mono-specific antibody may itself be labelled with an enzyme,fluorescent material, or radioactive material as described previously,and the presence of toxin A can be determined by detecting the presenceof this label. Alternatively, the mono-specific antibody bound to toxinA can be detected by use of labelled antibody specific for antibody ofthe animal in which the mono-specific antibody was raised; this binds tothe mono-specific antibody attached to toxin A. This method is referredto in the art as a double antibody sandwich form of the ELISA assay.

The presence of toxin A in the analyte may be determined by itsinteraction with mono-specific toxin A antibody in the assay.

The above procedure may also be employed for the determination of toxinB in an analyte by use of mono-specific antibody for toxin B in place ofmono-specific antibody for toxin A.

In another assay for toxin A of C. difficile, mono-specific antibody fortoxin A may be supported on a solid support; for example, a microtiterplate, and the supported mono-specific antibody for toxin A is contactedwith analyte suspected of containing toxin A, whereby any toxin Apresent in the sample (and only toxin A) becomes bound to the supportedmono-specific antibody. The presence and/or amount of bound toxin A maythen be determined by contacting the bound toxin A with C. difficileantibody, in labelled form, with such labelled antibody being bound byany bound toxin A. The presence and/or amount of toxin A present in theanalyte is then determined by determining the presence and/or amount ofthe bound labelled antibody.

The above procedure may also be used in an assay for toxin B bysubstituting mono-specific antibody for toxin B for the mono-specificantibody for toxin A.

In accordance with a further assay for toxin A, the analyte containingor suspected of containing toxin A, is contacted with a solid support,such as a microtiter tray so that at least the toxin A in the analyte issupported on the solid support. The presence of this toxin A is thendetected by mono-specific antibody for toxin A. The supported toxin Aselectively binds only the mono-specific antibody for toxin A. Thus, themono-specific antibody is supported on a solid support through thesupported toxin A of the analyte. This antibody can have a label, suchas an enzyme attached, that will allow its detection or a labelledantibody can be used that reacts with the antibody bound to the toxin A(sandwich ELISA method). The presence and/or amount of bound labelledantibody is a measure of the presence or amount of toxin A in theanalyte.

In accordance with a still further assay, toxin A may be detected by anagglutination procedure. According to such procedure, solid particlessensitized with mono-specific antibody to toxin A are contacted withanalyte containing or suspected of containing toxin A with the presenceof toxin A causing agglutination of such particles.

The agglutination assay is also suitable for detecting toxin B by usingmono-specific antibodies to toxin B in place of the mono-specificantibody to toxin A.

In accordance with still another assay, toxin A may be determined by aninhibition of agglutination procedure by contacting solid particlessensitized with purified toxin A (or sensitized with crude C. difficiletoxin, which includes toxin A) with both analyte containing or suspectedof containing toxin A, and mono-specific antibody for toxin A of C.difficile, with the presence of toxin A in the analyte inhibitingagglutination of the sensitized particles by the mono-specific antibody.Such procedure may also be employed for determining toxin B bysensitizing the particles with crude toxin and use of mono-specificantibody for toxin B.

As a further modification, the assay can be directed to determiningtoxigenic C. difficile (toxin A and/or toxin B) by use of antibody fortoxigenic C. difficile (a mixture of the mono-specific antibody fortoxin A and the mono-specific antibody for toxin B which is free ofdeterminant sites for non-toxic antigens). By using a mixture of suchmono-specific antibodies, it is possible to determine the presence ofeither toxin A or toxin B in a sample.

The present invention will be further described with respect to thefollowing examples; however, the scope of the invention is not to belimited thereby:

EXAMPLE I

This example is directed to the production of mono-specific antibody fortoxin A, and mono-specific antibodies for toxin B.

Bacteria and growth conditions

Two-liter brain heart infusion dialysis tube flasks were inoculated with0.1 ml of actively growing cultures of C. difficile VPI strain 11186(non-toxigenic) and C. difficile VPI strain 10463 (toxigenic), and theflasks were incubated at 37° C. for 3 days. The cells were obtained frominside the dialysis sack by centrifugation of the contents (9,000×g for15 minutes).

Preparation of boiled cell wash (BCW)-Sepharose, ToxinA-(ToxA)-Sepharose, and Toxin B(ToxB)-Sepharose

Strain 10463 packed cells (ca. 15 ml obtained from 12 flasks) werewashed 3 times (30 ml per wash) with 0.1M NaHCO₃ -0.5M NaCl, pH 8. Cellwashes were pooled and the pool was heated at 100° C. for 15 minutes.The precipitated material was removed by centriguation (12,000×g for 30minutes) and the supernatant fluid (ca. 34 mg. of protein in 90 ml) wasadded to 60 ml of Sepharose 4B (Pharmacia Fine Chemicals, Uppsala,Sweden) which had been activated with 18 g of CNBr. The suspension wasgently mixed at 4° C. overnight and uncoupled material was removed bywashing the gel with one bed volume of 0.1M NaHCO₃ -0.5M NaCl. Proteinanalysis of the wash indicated that the gel preparation contained ca.0.3 mg of protein per ml gel. The remaining active groups on theSepharose gel were blocked by adding one bed volume of 1M ethanolamine,pH 8, and mixing the gel at 4° C. overnight. The gel, designatedBCW-Sepharose, was washed 4× with alternating volumes (2 bed volumes perwash) of 0.1M sodium acetate-0.5M NaCl, pH 4, and 0.1M NaHCO₃ -0.5MNaCl, pH 8.

Partially purified toxin A and toxin B were prepared by ion exchangechromatography on DEAE Sepharose CL-6B (Pharamcia Fine Chemicals) asdescribed in Example II and each preparation was dialyzed overnight at4° C. against 0.1M NaHCO₃ -0.5M NaCl. Toxin A (ca. 3.3 mg of protein in20 ml) and toxin B (ca. 1.1 mg of protein in 20 ml) were each coupled,as described for BCW-Sepharose, to 20 ml of Sepharose 4B which had beenactivated with 7 g of CNBr. Protein analyses of the washes indicatedthat ToxA-Sepharose and ToxB-Sepharose contained 170 ug of protein and54 ug of protein per ml of gel, respectively.

Purification of monospecific antisera against Toxins A and B

Goat antiserum was prepared, as previously described, using refrigeratedformaldehyde (Ehrich, M., R. L. Van Tassell, J. M. Libby, and T. D.Wilkins, 1980. Production of Clostridium difficile antitoxin. Infect.Immun. 28:1041-1043.) against a crude C. difficile toxin preparationcontaining Toxins A and B. Antiserum (5 ml) was added to a suspension ofstrain 11186 cells (1.5 ml packed cells in 3 ml 0.85% NaCl) and themixture was gently homogenized with a Potter Elvehjam tissue grinder andthen rotated for 2 h at room temperature. The cells were subsequentlyremoved by centrifugation (12,000×g for 30 min) and the supernatantfluid was passed through a 0.45 um membrane and concentrated to 1× witha minicon-B15 concentrator (Amicon Corp, Lexington, Mass.). Strain 11186cell-adsorbed antiserum (4.1 ml) was applied to a column (1.5 by 31.4cm) of BCW-Sepharose, and nonadsorbed material was eluted at roomtemperature with 2 bed volumes of 0.1M NaHCO₃ -0.5M NaCl, pH 8, at aflow rate of 40 ml/h. The eluate was concentrated to 1× byultrafiltration in a stirred cell equipped with a PM 10 membrane (AmiconCorp.). The BCW-Sepharose-eluate (4.1 ml) was divided into 2 equalportions which were applied to columns (1 by 25 cm) of ToxA-Sepharoseand ToxB-Sepharose. Nonadsorbed material was eluted at room temperaturefrom each column with 2 bed volumes of 0.1M NaHCO₃ -0.5M NaCl, pH 8, ata flow rate of 40 ml/h. Eluates were concentrated to 1× byultrafiltration.

Elution of antibodies bound to ToxA-Sepharose and ToxB-Sepharose

Following the elution of nonadsorbed material from ToxA-Sepharose andToxB-Sepharose, the columns were washed with 0.1M NaHCO₃ -0.5M NaCl., pH8, until there was no measurable adsorbance at 280 nm. Antibodies boundto the gels were eluted by applying 5 ml of 3.5M KSCN, pH 6.8, to eachcolumn and washing with 0.1M NaHCO₃ -0.5M NaCl. Approximately 2 bedvolumes were collected from each column. The eluates were dialyzedagainst 4 l of 0.1M borate-buffered saline pH 8.5, at 4° C. overnightand concentrated to 1× by ultrafiltration.

The antibody eluted from the ToxA-Sepharose column is the mono-specificantibody for toxin A of C. difficile and the antibody eluted from theToxB-Sepharose column is the mono-specific antibody for toxin B of C.difficile.

Purification of IgG fraction

The eluted antibodies from the ToxA-Sepharose and ToxB-Sepharose columnwere purified by chromatography on DEAE Affi-Gel Blue (Bio-RadLaboratories, Rockville Centre, NY) as recommended by the manufacturerfor the purification of rabbit IgG. Antiserum samples (2 ml) wereapplied to a column of DEAE Affi-Gel Blue (1 by 31.8 cm) and eluted at aflow rate of 20 ml/h. Fractions (2 ml) containing purified IgG werepooled and concentrated to 1× by ultrafiltration.

EXAMPLE II

This example is directed to production of pure toxin A of C. difficile.

Bacterial strain. Clostridium difficile VPI strain 10463 was grown intwo liter brain heart infusion (BHI) dialysis flasks for 72 hours at 37°C. After centrifugation at 8000×g for 10 minutes and filtration througha 0.45 um membrane filter (Millipore Corp., Bedford, MA), the culturesupernatant (c.750 ml) was concentrated to 50 ml by ultrafiltration, at4° C., using an XM-100 membrane filter (Amicon Corp., Lexington, MA)with a thin channel type concentrator. The retentate was washed with1500 ml of 50 mM TRIS-HCl buffer, pH 7.5 (4° C.) and concentrated to afinal volume of 40-50 ml. This removed many small molecular weightcontaminants. The concentrated supernatant was loaded onto a 2.5 by 10cm DEAE Sepharose CL-6B column which had been equilibrated with 50 mMTRIS-HCl, pH 7.5. After sample loading, the column was washed with 200ml of 50 mM TRIS-HCl, pH 7.5, containing 0.05M NaCl. The sample waseluted first with a 300 ml linear NaCl gradient in 50 mM TRIS-HCl buffer(0.05-0.25M NaCl), followed by 150 ml of 50 mM TRIS-HCl, pH 7.5,containing 0.3M NaCl. A second 300 ml linear gradient (0.3-0.6M NaCl) inthe same buffer followed the 0.3M NaCl wash. The flow rate of thecolumns was 55-60 ml per hr (Gravity) at 4° C. Fractions (4.2 ml) werecollected and assayed for cytotoxicity using CHO-Kl cells.

The fractions containing the highest cytotoxic titers were pooled,filter-sterilized and stored at 4° C. The toxins that eluted in thefirst and second NaCl gradients were designated Toxins A and Brespectively, and are partially purified toxins A and B, respectively.

Five to ten ml of the toxic fractions from the first DEAE gradient(Toxin A) were dialyzed against one liter of 0.01M sodium acetate bufferpH 5.5 at 4° C. for 18-24 hours. The dialysate was centrifuged torecover the precipitate at 169×g for 10 minutes and was then washed with5 ml of the same acetate buffer and centrifuged again. The precipitatewas solubilized in 5-10 ml of 50 mM TRIS-HCl, pH 7.5 containing 0.05MNaCl and the solution of purified toxin A was filter-sterilized andstored at 4° C.

EXAMPLE III

The following buffers are used in an assay for Toxins A and B.

Carbonate buffer (coating buffer)

1.59 g Na₂ CO₃

2.93 g NaHCO₃

0.20 g NaN₃

bring to 1 liter with dH₂ O; pH 9.6;

store at room temperature (use within 2 weeks)

Phosphate-buffered saline-Tween 20 (PBS-T)

8.0 g NaCl

0.2 g KH2PO₄

2.9 g Na₂ HOP₄ ·12H₂ O (2.2 g Na₂ HOP₄ ·7H₂ O)

0.2 g KCl

0.5 ml Tween 20 (polyoxyethylene sorbitan monolaurate)

0.2 g NaN₃

bring to 1 liter with dH₂ O; pH 7.4;

Diethanolamine buffer (for alkaline phosphatase substrate)

97 ml diethanolamine

800 ml dH₂ O

0.2 g NaN₃

100 mg MgCl₂ ·6H₂ O

titrate to pH 9.8 with 1M HCl and bring volume to 1 liter with dH₂ O;store in dark bottle at room temperature; for substrate solution, add 1mg substrate per ml buffer;

Assay for Clostridium difficile Toxins A and B

(1) Add 200 ul of 1/10,000 dilution (in carbonate buffer, pH 9.6) ofrabbit antiserum (antibody to C. difficile) to each well of a DynatechImmulon type 2 microtiter plate. Incubate at 4° C. overnight.

(2) Empty plate and add 200 ul of PBS-T containing 0.5% bovine serumalbumin to each well. Incubate plate at 37° C. for 30 minutes.

(3) Empty plate and add 200 ul of PBS-T to each well. Incubate plate atroom temperature for 5 minutes.

(4) Empty plate and add 200 ul of sample dilution or toxin dilution(1:2) in PBS-T to wells. Incubate plate either at 37° C. for 1 hour orat room temperature overnight.

(5) Empty plate and wash each well 3 times with PBS-T.

(6) Add 200 ul of 1/1,000 dilution in PBS-T of monospecific antibody foreither Toxin A or Toxin B to each well. Incubate plate at 37° C. for 1hour.

(7) Empty plate and wash each well 3 times with PBS-T.

(8) Add 200 ul of 1/800 dilution (in PBS-T) of rabbit antigoat IgGcoupled to alkaline phosphatase to each well. Incubate plate at 37° C.for 1 hour.

(9) Empty plate and wash each well 3 times with PBS-T.

(10) Add 200 ul of p-nitrophenylphosphate (1 mg/ml in diethanolaminebuffer) to each well. Incubate plate at room temperature for 1 hour.

(11) Add 20 ul of 5N NaOH to each well to terminate the reaction.

(12) Mix contents of each well with 0.8 ml dH₂ O (total volume of assaymixture ca. 1 ml) and measure the absorbance at 405 nm.

The present invention is particularly advantageous in that it ispossible to produce antibodies which are specific for only the toxins ofC. difficile. As a result, there is provided an assay which is directedto determining the presence of these toxins, rather than C. difficile,which will reduce or eliminate false positives.

Furthermore, the present invention offers the advantage of permitting anassay which can be directed to either of the toxins or both toxins.

An assay for the toxins in accordance with the invention is rapid andalso less costly than prior assays.

Numerous modifications and variations of the present invention arepossible in light of the above teachings, and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as specifically described.

What is claimed is:
 1. A process of preparing pure toxin A of C.difficile comprising:Adjusting the pH and molarity of an aqueoussolution of partially purified toxin A of C. difficile, which issubstantially free of toxin B and containing some nontoxigenic proteins,whereby the toxin A is precipitated without precipitation ofnontoxigenic proteins and without denaturation of the toxin A, said pHbeing at a value of less than 6.0, said molarity being less than 0.1M;and recovering the precipitated toxin A as pure toxin A.
 2. The processof claim 1 wherein the pH is at least 5.0 and the molarity is at least0.001M.
 3. The process of claim 2 wherein the pH is from 5.3 to 5.7. 4.The process of claim 3 wherein the pH is 5.5 and the molarity is 0.01M.